The development of erythroid cells from multipotential progenitors is regulated by transcription factors that coordinate gene expression during cellular growth and maturation. Characterization of these DNA binding proteins and their target genes has been helpful in elucidating the molecular basis of normal hematopoietic differentiation, a process that goes awry in acquired and inherited blood disorders such as leukemias and thalassemias. Gene knockout studies in mice have established that the related transcription factors GATA-I and GATA-2 are essential for normal erythropoiesis. However, the precise roles for these DNA-binding proteins during erythroid ontogeny, and the specific GATA-target genes required at different stages of red cell development, have been difficult to define by in vivo studies. The experiments described here address these problems, primarily through in vitro hematopoietic differentiation of GATA-I gene disrupted embryonic stem (ES) cells, an approach that provides pure GATA-1 negative erythroid precursors for morphologic and molecular analysis. Preliminary studies identify a maturation arrest followed by apoptosis in GATA-1 negative proerythroblasts, despite normal levels of mRNA encoding numerous GATA target genes including globins and the erythropoietin receptor. Moreover, the observation that GATA-2 is upregulated some 50-fold may explain,the transcription of these GATA targets, but GATA-2 is not permissive for terminal erythropoiesis. Future experiments will focus on the identification of unique GATA-I and GATA-2 target genes in erythroid cells, and the mechanisms that lead to apoptosis in erythroblasts lacking GATA-I. To facilitate these studies, a GATA-I negative erythroid cell line will be developed and characterized.